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KCl 2.7 mM, KH 2 PO 4 1.5 mM, NaCl 136 mM, Na 2 HPO 4 2 H 2 O 6.5 mM, pH 7.4) containing 3% bovine serum albumin (BSA-PBS) for 1 h at room temperature and were then incubated with a rabbit anti-claudin 5 primary antibody diluted in 3% BSA-PBS (1:300) overnight at 4 C